Nanobodies targeting EGFR provide insight into conformations stabilized by glioblastoma mutations.

Oncogenic mutations in the epidermal growth factor receptor (EGFR) promote tumorigenesis by stabilizing active or pre-active receptor conformations. Most EGFR-driven cancers are characterized by kinase domain mutations that directly activate the receptor. However, brain cancers such as glioblastoma multiforme (GBM) uniquely harbor mutations in the EGFR ectodomain that allosterically activate the kinase domain. Despite significant advances in understanding the physiological and pathogenic roles of EGFR, the conformational characteristics that define ligand-independent EGFR activation in GBM remain poorly understood. In this study, we use naive and post-immune yeast-displayed nanobody libraries to discover four nanobody groups that with benchmark nanobodies define a total of five groups with unique binding signatures and specificities for GBM mutation-stabilized conformational states. Nanobodies in Groups 1 and 2 block ligand, selectively bind the inactive, tethered conformation, and favor wild-type EGFR over GBM-stabilized conformations. In contrast, nanobodies in Groups 4 and 5 do not block ligand, target active or pre-active conformations, and selectively bind GBM-stabilized conformations. Additionally, nanobodies in Group 3 block ligand and appear to be conformation agnostic. We observed domain-specific bias in the nanobodies' selectivity for GBM mutations, suggesting that mutations across different ectodomain regions stabilize distinct conformations. This work advances our understanding of EGFR conformational equilibria in the context of GBM. The observed cooperativity and mutation-dependent binding of nanobodies emphasize their utility in dissecting EGFR activation mechanisms and in developing targeted therapies for EGFR-driven cancers, including GBM.